Process to control the molecular weight and polydispersity of substuted polyphenols and polyaromatic amines by enzymatic synthesis in organic solvents, microemulsions, and biphasic systems

ABSTRACT

A process of controlling the molecular weight and dispersity of poly(p-ethylphenol) and poly(n-cresol) synthesized enzymatically by varying the composition of the reaction medium. Polymers with low dispersities and molecular weights from 1000 to 3000 are synthesized in reversed micelles and biphasic systems. In comparison, reactions in bulk solvents resulted in a narrow range of molecular weights (281 to 675 with poly(p-ethylphenol) in a DMF/water system and 1,400 to 25,000 with poly(m-cresol) in an ethanol/water system). Poly(p-ethylphenol) was functionalized at hydroxyl positions with palmitoyl, cinnamoyl, and biotin groups.

STATEMENT OF GOVERNMENT INTEREST

[0001] The invention described herein may be manufactured and used bythe Government for governmental purposes without the payment of anyroyalty thereon.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the preparation ofphenolic and aromatic amine polymers, wherein the reaction conditionsare controlled such that high product yields, molecular weight, and auniform molecular weight distribution are obtained.

BACKGROUND OF THE INVENTION

[0003] Phenolic and aromatic amine polymer resins constitute a veryimportant and useful class of chemical compounds. They have a number ofuses, e.g., as coatings and laminates that provide a number offunctional advantages. Besides possessing good thermal properties, thesepolymers can be doped to make them electrically conductive, making thema key component of integrated circuit (IC) chips.

[0004] At present, these polymers are prepared by chemical synthesis,e.g., as from phenol and formaldehyde. The polymers's linearity/networkstructure (and, by extension, their functional properties) variesdepending on the monomer and type of reaction conditions used. However,the use of certain constituent chemicals, such as formaldehyde, is beingrestricted in the chemical industry because of their toxicity.Accordingly, the enzyme-mediated synthesis of polyphenols andpolyaromatic amines offers a viable alternative to the currently usedchemical synthesis of such commercial phenolic resins.

[0005] Peroxidase-catalyzed free radical polymerization of phenol,aromatic amines, and their derivatives is well known. Horseradishperoxidase (HRP) is the most widely used biocatalyst in thepolymerization of phenol, aniline, or their derivatives. HRP has beenshown to be active in a number of organic solvents or solvent mixturesand the reaction is typically initiated by the addition of hydrogenperoxide as an oxidant.

[0006] Dordick et al., Vol. # 30 1987 Biotechnol. Bioeng. 31-36, usedHRP in a dioxane/water system to prepare a number of polymers andcopolymers from various phenolic monomers. Akkara et al., 29 J. Poylm.Sci. A 1561 (1991), prepared polymers and copolymers of various phenolsand aromatic amines using these same reactions and carried out detailedcharacterization of the polymer products. p-Alkylphenols were alsopolymerized at oil-water (reversed micelles) and air-water(Langmuir-Blodgett trough) interfaces. Because of their amphiphilicnature, the alkylphenols are positioned at the interface, and in thepresence of HRP and hydrogen peroxide the monomers are oxidativelycoupled to form polymers. The poly(p-alkylphenols) prepared in reversedmicelles were shown to exhibit relatively more uniform molecular weightdistribution than those prepared in bulk organic solvents.

[0007] However, earlier attempts to control the polymer molecular weightand molecular weight distribution by varying the time of reaction orhydrogen peroxide concentration were unsuccessful in both reversedmicelles and bulk solvents. Initial hydrogen peroxide concentration wasfound to be stoichiometrically proportional to the monomer conversion, ahallmark of stepwise polymerization and a phenomenon observedpreviously, and there was no effect on the polymer molecular weight andpolydispersity.

[0008] The polymers can be modified by adding functional groups to thepolymeric backbone, significantly enhancing the utility of thesepolymers. “Functionalization” enables the polymers to be used to treatfabrics, to form selectively permeable membranes, and to improve theperformance of IC chips, among other applications.

[0009] Palmitoyl chlorides may be added to the polymer to make thepolymer easily processable, e.g., as coatings, films, or finishes.Cinnamoyl chloride may be added to create controlled pore size membranes(e.g. , “molecular sieves”) or to enhance the polymers's ability toabsorb UV radiation (e.g., for sunglasses), thereby enabling their useas anti-reflective coatings in photoresists. In their latter use, themodified polymers are applied to a silicon substrate as an undercoating(under non-functionalized polyphenols or polyaromatic amines that arethen applied as a spin coating) in an IC chip to control the precisionof UV etching, by inhibiting UV scattering, of circuitry into thespin-coated polymer layer. In addition, these cinnamoylchloride-modified polymers are very thermostable, which allows their usein a variety of applications where heat is ordinarily a problem. Incontrast, photosensitive functional groups may be added to enhance theutility of the polymers in other applications.

[0010] The polymers also may be modified to create active matrices andsystems allowing the controlled-release of materials, such as drugs,insecticides, and fertilizers. If biotin groups are added to the polymerchain, the polymer can be used as chromatography packing, which may beused to separate and purify proteins.

[0011] Despite the study of how the functionality of the polymers variesdepending upon whether, and with what, the molecules are modified, ithas not been shown that the molecular weight and the molecular weightdistribution (i.e., the “polydispersity”) of polyphenols andpolyaromatic amines also can significantly influence the functionalproperties of the polymers.

[0012] Accordingly, it is an object of this invention to overcome theabove illustrated inadequacies and problems of extant polyphenols andpolyaromatic amines by providing an improved method of their manufacturesuitable for use in applications that would benefit from uniform polymersize.

[0013] It is another object of this invention to provide a method ofproducing polyphenols and polyaromatic amines wherein it is possible tocontrol the molecular weight distribution of the polymer molecules.

[0014] Yet another object of the present invention is to provide amethod of producing polyphenols and polyaromatic amines wherein themolecular weight distribution of the polymer molecules is between 600and 3,600.

[0015] It is a further object of the present invention to provide amethod of producing polyphenols and polyaromatic amines wherein themolecular weight distribution of the polymer molecules is between 1,400and 25,000.

[0016] A still further object is to provide a method of producingpolyphenols and polyaromatic amines wherein it is possible to controlthe polydispersity of the polymer molecules.

[0017] It is another object of this invention to provide a method ofproducing polyphenols and polyaromatic amines wherein the polydispersityof the polymer molecules ranges from 1.02 to greater than 2.

[0018] It is yet another object of the present invention to provide amethod to modify the polymer prepared by adding functional groups to thepolymer using palmitoyl chloride, cinnamoyl chloride, and biotincompounds.

SUMMARY OF THE INVENTION

[0019] The objects of the present invention are met by a method ofenzymatically synthesizing polyphenols and polyaromatic amines undercontrolled reaction conditions. More particularly, the invention relatesto the control of molecular weight and polydispersity in enzymaticallysynthesized polyphenols and polyaromatic amines by manipulating theseveral reaction parameters.

[0020] The present invention defines reaction conditions for any givenphenol/aromatic amine monomer necessary to control M_(w) andpolydispersity within a defined range. Such control of M_(w) andpolydispersity has been found to increase the utility of these polymers.

[0021] In particular, the ability to control the molecular weight anddispersity of poly(p-ethylphenol) and poly(m-cresol) has been achieved.The polymers were synthesized enzymatically in different organicsolvents and a water-in-oil microemulsion. Using solubility parameters,the composition of the reaction medium was varied to study the effectson polymer yield, molecular weight, and dispersity. It has beendiscovered that polymers with low dispersities and with molecularweights ranging from 1000 to 3000 can be synthesized in reversedmicelles. In addition, it has been discovered that reactions conductedin bulk solvents resulted in a narrow range of molecular weights (281 to675 with poly(p-ethylphenol) in a dimethylformamide (DMF)/water systemand 1,400 to 25,000 with poly(m-cresol) in an ethanol/water system).

[0022] With DMF as the chromatography eluent, the effect of LiBr in DMFon the molecular aggregation of poly(p-ethylphenol) was determined usinggel permeation chromatography (GPC). The presence of LiBr (at 0.35 w/v%) in DMF resulted in complete dissociation of the aggregates insolution. Further, poly(p-ethylphenol) was functionalized at hydroxylpositions with palmitoyl and cinnamoyl groups. Structuralcharacterization of the polymers was carried out by ¹³C-NMR, UV, andFTIR spectroscopies.

[0023] Other objects, features and advantages will be apparent from thefollowing detailed description of preferred embodiments thereof taken inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 is a schematic of ortho- and para-substituted phenolpolymerization catalyzed by horseradish peroxidase (HRP);

[0025]FIG. 2 is the ¹³C-NMR spectra for (a) p-ethylphenol and (b)poly(p-ethylphenol);

[0026]FIG. 3 is a graph of the effect of LiBr concentration in DMF onpoly(p-ethylphenol) molecular weight;

[0027]FIG. 4a is a differential scanning calorimetry (DSC) thermogram ofpoly(p-ethylphenol) prepared in reversed micelles;

[0028]FIG. 4b is a thermogravimetric analysis (TGA) of p-ethylphenol andpoly(p-ethylphenol) prepared in reversed micelles;

[0029]FIG. 5 shows the molecular weight distribution ofpoly(p-ethylphenol) before and after heating;

[0030]FIG. 6a shows FTIR spectra of poly(p-ethylphenol) (i) before and(ii) after esterification with palmitoyl chloride;

[0031]FIG. 6b shows FTIR spectra of poly(p-ethylphenol) (i) before and(ii) after esterification with cinnamoyl chloride; and

[0032]FIG. 7 shows the UV absorbance at 259 nm of poly(p-ethylphenol)before and after cinnamoylation;

DETAILED DESCRIPTION OF EMBODIMENTS

[0033] Free radical polymerization of p-ethylphenol and m-cresol,catalyzed by horseradish peroxidase, was carried out at ambientconditions in a number of organic solvent systems. While theAOT/isooctane reversed micellar system afforded complete monomerconversion into polymer with an average molecular weight of 2,500, theaddition of chloroform yielded lower molecular weights, with narrowerdistributions. Reactions carried out in DMF produced mostly oligomerswith uniform molecular weights. Poly(m-cresol) molecular weight could becontrolled between 1,400 and 25,000 by appropriate design of thereaction medium comprised of ethanol-water mixture. Analysis ofpoly(p-ethylphenol) by GPC demonstrated the effect of LiBr on themolecular weights of poly(p-ethylphenol) and poly(p-phenylphenol). Thepolymers showed apparently high molecular weights in DMF as GPC solventdue to significant inter/intra-molecular associations. At 0.35% LiBr inDMF and above, these associations were eliminated to permit theestimation of true molecular weights. ¹³C-NMR and FTIR studies revealedthat the repeat units in poly(p-ethylphenol) are primarily linked atortho positions. The hydroxyl groups, which are not involved in bondformation, could be derivatized with palmitoyl and cinnamoyl chlorides.

Example 1

[0034] A typical polymerization reaction was carried out in reversedmicelles as follows. A 10 ml solution of 0.15 M dioctyl sodiumsulfosuccinate (AOT) in isooctane was prepared, and 0.4 ml of an aqueouspreparation of horseradish peroxidase (Type II) (12.5 μM) was added toform a clear reversed micellar solution having a W_(o) (molar ratio ofwater to surfactant) of about 15. p-Ethylphenol was added to thereversed micellar solution, and the polymerization reaction wasinitiated by adding drops of 30% hydrogen peroxide (w/w) (up to about30% stoichiometric excess) while stirring the reaction mixture.

[0035] The reaction was exothermic with rapid formation of a yellowishprecipitate After continuing the stirring for several hours, theprecipitate was centrifuged and washed repeatedly with pure isooctane toremove the surfactant and any unreacted monomer. The final precipitatewas dried overnight under a reduced pressure at 50° C.

Example 2

[0036] In cases where a mixture of chloroform and isooctane was used toform reversed micelles, the same procedure was followed except that thecorresponding solvent mixture was used in place of isooctane. Isooctaneand chloroform were stored with molecular sieves to remove water fromthe solvents. However, stable (i.e., transparent and single-phase)reversed micellar solutions were found to be difficult to form with amixture of chloroform and isooctane with 25% or less isooctane. A stablemicroemulsion could be obtained only up to a W_(o) of 9 with 50%chloroform at room temperature, and phase separation occurred at highervalues.

Example 3

[0037] In the absence of reversed micelles, reaction mixtures wereprepared by first dissolving the monomer and the enzyme in a mixture ofHEPES (N-[2-Hydroxyethyl]piperazine-N′[2-ethanesulfonic acid]) bufferand solvent such as N,N-dimethylformamide (DMF). The reaction wasinitiated, as before, by the dropwise addition of hydrogen peroxide. Theenzyme was completely soluble at 0.5 mg/ml concentration in DMF/watermixtures at all solvent compositions studied.

[0038] A. Structural Characterizattion

[0039]FIG. 1 illustrates the reaction scheme and the structures ofmonomers used. Crosslinking in polymer structure is expected in thosecases where the ortho and para positions in the corresponding monomerstructure are unsubstituted, as is the case with p-phenylphenol andm-cresol. As shown in FIGS. 2a & 2 b, ¹³C-NMR studies onpoly(p-ethylphenol) indicate that the linkage between any two adjacentphenyl rings is largely at the ortho positions. However, this type oflinkage may strain the polymer backbone in such a manner that the phenylrings are out of plane with respect to the adjacent rings. As a result,the polymer backbone may be forced into a coiled structure.

[0040]¹³C-NMR spectra on the monomer and polymer were recorded on a 200MHz Varian instrument (C broad band probe, Model XL-200, Palo Alto,Calif.). Deuterated acetone and tetramethylsilane (TMS) were used as thesolvent and the internal standard, respectively. Infrared spectra wererecorded on a Perkin-Elmer 1760 FTIR-FTRaman spectrophotometer at 4 cm⁻¹resolution. The samples were cast as thin films on a KBr window fromchloroform solutions. UV spectroscopy studies were carried out on aBeckman DU 7500 spectrophotometer.

[0041]FIGS. 2a & 2 b illustrate ¹³C-NMR spectra and peak assignments forthe monomer and the polymer, respectively. The peak for C2 & C6 (at115.9 ppm in the monomer and 117 ppm in the polymer) diminished while anadditional peak appeared at 131 ppm in the polymer. The peak position at131 ppm is in agreement with the theoretically calculated peak positionfor ortho linkages on the ring. On the other hand, if the monomer werelinked at meta positions on the ring, the peaks for C3 and C5 shouldshlift downfield from 129.4 ppm in the monomer to 144 ppm in thepolymer. However, the polymer spectrum in FIG. 2b shows no such peak,therefore ruling out linkages at meta positions. There was nosignificant change in the peak position for C4, therefore ruling outether linkages. Although the hydroxyl groups are involved in theformation of free radicals leading to polymer formation, they do notappear to be involved in bond formation. In addition, previous infraredstudies revealed no ether linkage in the polymer structure. Thus thephenyl rings in the polymer appear to be linked primarily at orthopositions. The presence of free hydroxyl groups is also indicated byFTIR [see FIG. 6a(i) and 6 b(i)].

[0042] B. Molecular Weight Determination

[0043] Molecular weights were determined on a Waters LC Module Iinstrument with an on-line GPC column (GBR mixed bed linear column witha molecular weight range of 100 to over 20 million). A UV detector at270 nm was used to detect the polymer. The GPC data were collected andprocessed with Millennium GPC software supplied with the instrument. Aneluent flow rate of 1 ml/min was maintained under isocratic conditions.Narrow molecular weight polystyrene standards were used for calibration.All samples were filtered through 0.2 micron PTFE filters prior toinjection. It was ascertained that the filters did not retain anypolymer during filtration.

[0044] The effects of LiBr in DMF on aggregation phenomena, as reflectedby the weight average molecular weight of poly(p-ethylphenol), were alsodetermined. LiBr is used to get true chromatographic separation basedupon M_(w) (LiBr breaks apart aggregated polymer molecules). A precisemeasure of M_(w) is necessary to determine the functional utility ofpolymer.

[0045] For GPC analysis, poly(p-ethylphenol) was completely dissolved ata concentration of 1 mg/ml in a series of DMF-based solutions withvarying LiBr concentrations in the range of 0 to 1% (w/v). A givencomposition (between 0 and 1% LiBr/DMF) of the GPC solvent was preparedby mixing pure DMF and 1% LiBr/DMF in appropriate proportions. For allinjections, the composition of the GPC solvent and the solvent used toprepare the sample for injection were identical. A mixture ofpolystyrene standards (M_(w) 122 to 2.7 million, narrow distributionwith polydispersity in the range of 1.02 to 1.2) was prepared in allcompositions of LiBr and DMF, and always injected before analyzing thepolyphenol sample in the corresponding solvent.

[0046] Dimethylformamide is a good solvent for solution studies ofpolyphenols. Earlier reports used a mixture of DMF and methanol, at aratio of 4 to 1, as a GPC solvent in the determination of molecularweights of polyphenols. DMF is an interesting solvent, especially forpolyhydroxy compounds such as polysaccharides and polyphenols. Forexample, amylose is not soluble is DMF, but the polysaccharide swells asDMF penetrates into and ‘wets’ the polymer. However, it is well knownthat in the presence of about 3% (w/v) LiBr, amylose could be dissolvedat a concentration of about 1% (w/v) in DMF. Polyphenol like apolysaccharide, is also a polyhydroxy compound. Although DMF easilydissolves poly(p-ethylphenol), there still may be inter/intramolecularassociations in the polymer. These interactions may result in anapparently high molecular weight in GPC analysis.

[0047] The potential aggregation of polyphenol molecules, and the use ofa mixture of DMF and methanol to break the association, is known.molecular weights in the range of a few hundreds to a few thousands havebeen reported for a number of different polyphenols, withpoly(p-phenylphenol) exhibiting a molecular weight of 26,000. Using anidentical GPC solvent composition, molecular weights of over 400,000 forpoly(p-phenylphenol) prepared in a dioxane/water system have beenreported. Similarly, an average molecular weight of about 20,000 withDMF/methanol solvent mixture as GPC eluent for poly(p-ethylphenol)prepared in AOT reversed micelles has been observed. However, it is notclear if the solvent mixture of DMF and methanol at 4:1 ratio is optimalto deaggregate the polymer chains and give a true molecular weight. Toaddress this problem, the molecular weights of poly(p-ethylphenol)prepared in reversed micelles and dioxane/water system andpoly(p-phenylphenol) prepared in dioxane/water system were analyzed as afunction of LiBr concentration in DMF and DMF/methanol mixture as GPCeluents.

[0048]FIG. 3 illustrates the effect of LiBr concentration in DMF as GPCeluent on the weight average molecular weight of poly(p-ethylphenol)prepared in reversed micelles. There is a dramatic decrease in themolecular weight by over three orders of magnitude when the LiBrconcentration was increased from 0 to 0.35%. The molecular weight anddispersity of the polymer stabilized at about 2500 and 1.36,respectively, above 0.35% LiBr in DMF. Above this critical concentrationof LiBr in DMF, there is no additional effect on the polymer a molecularweight.

[0049] An analogous phenomenon was observed with the solubility studiesof amylose in DMF. Although DMF is capable of forming its own hydrogenbonds with the polysaccharide (as noted earlier, the polysaccharideswells in DMF, but is insoluble), it may not be able to completelydisrupt the intermolecular forces. However, LiBr appears to be veryeffective in overcoming these intermolecular interactions. Thepolysaccharide becomes soluble at a concentration of 3% LiBr in DMF. Itis possible that the solubility of the polyhydroxy compound is dictatedby a fixed ratio between amylose and LiBr concentrations in DMF. Thesame argument applies to the molecular dissociation ofpoly(p-ethylphenol) in the presence of LiBr in DMF. Not unexpectedly,there was no effect of LiBr on the retention times of the polystyrenestandards due to the lack of strong interchain interactions.

[0050] A mixture of DMF/methanol at 4:1 ratio was also used as the GPCsolvent to determine the molecular weight of poly(p-ethylphenol)synthesized in AOT/isooctane reversed micelles. The result was a bimodaldistribution with an average molecular weight of 90,000 and 300,000 forthe two distributions. A similar bimodal molecular weight distributionwas described by Akkara et al. for poly(p-phenylphenol). Molecularaggregation is still significant in this solvent system since themolecular weight of the sample dropped to about 2700 in the presence of1% LiBr in DMF/methanol mixture at 4:1 ratio. Identical observationswere made with a sample of poly(p-ethylphenol) synthesized in 85%dioxane/water system. Subsequently, a sample of poly(p-phenylphenol),synthesized in 85% dioxane/water, was analyzed for molecular weight bothin DMF and DMF/methanol mixtures at different LiBr concentrations. Asbefore, the polymer molecular weight dropped from well over 6 million toabout 3400 on increasing LiBr concentration from 0 to 1% (w/v) in DMF.Similarly, poly(p-phenylphenol) showed a significant shift to lowermolecular weight as the LiBr concentration in DMF/methanol mixture at4:1 ratio was varied in the same concentration range as in DMF. In thiscase, the molecular weight dropped from about 500,000 to 3200. Table 1lists the molecular weight and dispersity profiles ofpoly(p-ethylphenol) and poly(p-phenylphenol) synthesized under differentconditions as a function of GPC solvent composition. Poly(p-ethylphenol)synthesized in reversed micelles exhibited a polydispersity of less than1.4, and that prepared in bulk solvent, dioxane/water, >2. The averagemolecular weight of the polymer increased slightly as the surfactantconcentration was increased, a phenomenon noted earlier. TABLE 1Molecular weight and dispersity profiles of poly(p- ethylphenol) andpoly(p-phenylphenol) synthesized under different conditions as afunction of GPC solvent composition. Synthesis M_(w)(M_(w)/M_(n))¹Sample medium (a) (b) (c) (d) Poly(p- AOT/isooctane >4.5M 2500 300,0002700 ethylphenol) reversed micelles (>2.5)  (1.4) (>2.0)  (1.4) Poly(p-85/15 >6.0M 3400 500,000 3200 ethylphenol) dioxane/water (>2.5) (>2.0)(>2.0) (>2.0) Poly(p- 85/15 >6.OM 3000 300,000 3200 phenylphenol)dioxane/water (>2.5) (>2.0) (>2.0) (>2.0)

[0051] C. Thermal Characterization

[0052] Thermal characterization of polymers was carried out on Du Pontthermal analyzers. For differential scanning calorimetry (DSC) analysis,the polymers were hermetically sealed, and heated under a nitrogenatmosphere at a temperature gradient of 10° C. per minute from roomtemperature to 300° C. Thermogravimetric analysis (TGA) was carried outat the same temperature gradient and under nitrogen atmosphere, butheated to 600° C.

[0053] The thermal properties of p-ethylphenol and poly(p-ethylphenol)prepared in reversed micelles are illustrated as DSC and TGA thermogramsin FIGS. 4a & 4 b, respectively. The polymer was reasonably stable untila temperature of about 250° C., with a loss of less than 10% of thematerial (6% loss occurred at 200° C. presumably in part due to loss ofwater). The exotherm at about 110° C. in the polymer DSC thermogram maybe due to cross linking in the polymer or due to loss of heat ofcrystallization. Once heated over 200° C., the exotherm was irreversiblylost.

[0054] A 170% increase in molecular weight was observed, presumably dueto cross-linking, when a sample of poly(p-ethylphenol) was heated to150° C., and the polymer became significantly more polydisperse than thecorresponding untreated polymer. FIG. 5 shows portions of the GPCprofiles of poly(p-ethylphenol) before and after heating the polymer to150° C. Both samples were easily soluble in 1% LiBr/DMF solution thatwas used as eluent. X-ray diffraction studies on the samples revealed apartial crystallization of the heat-treated polymer.

Example 4

[0055] Phenol polymerization was carried out in a mixture of DMF andwater at various ratios to investigate the solvent effect on enzymeactivity and on the polymer molecular weight. The objective was toinvestigate if the molecular weight of polyphenols could be controlled,while maintaining a reasonably narrow distribution, by varying reactionsystem parameters such as time of reaction, hydrogen peroxideconcentration, and solvent composition. Table 2 sets forth the ranges ofsolubility parameters and dielectric constants covered by the solventsystems used for polymerization reactions. TABLE 2 Ranges of solubilityparameters and dielectric constants covered by the solvent systems usedfor polymerization reactions. Solubility parameter Dielectric Solventsystem (MPa^(½)) constant Isooctane/ 14  2 chloroform 19  5 DMF/ 25 37water 48 78 1,4-Dioxane/ 20 30 water 48 78 Ethanol/ 26 24 water 48 78

[0056] The solvent mixtures, listed in Table 2, were selected on thebasis of the range of solubility parameters that they cover. The widevariation in solubility parameters and dielectric constants for eachsystem was similar to that found in certain supercritical fluids as afunction of pressure. These properties not only influence the solubilityof the growing polymer chain in the corresponding reaction medium, butsignificantly affect enzyme activity (solvents with high dielectricconstants are known to denature the enzyme). However, unlike insupercritical fluids, the solvent properties can be varied at ambientconditions of pressure and temperature.

[0057] The reaction medium composition was varied from 100% DMF to 100%water. As before, the reaction was initiated with the addition ofhydrogen peroxide at room temperature and with stirring. Interestingly,and analogous to the dioxane/water system, there was no sign of reaction(i.e., no heat or color generation) in the reaction mixtures containing85% or more organic solvent, and the solutions remained clear throughoutthe addition of hydrogen peroxide. On the other hand, heat evolution(due to exothermic reaction) followed the reaction in solvents with 60%or less DMF, and the solutions became colored and opaqueinstantaneously.

[0058] It is clear that DMF sustained enzyme activity up to aconcentration of 60%, although the presence of water was necessary. Themonomer solubility in 20% DMF solution was poor, and solution turnedinto a stable emulsion prior to initiating the reaction. The reactionswere continued for a few more hours before the solvent was evaporatedunder reduced pressure. The precipitates were washed with water andisooctane to remove buffer salt, the enzyme and any unreacted monomer.The dried precipitates were dissolved in 1% LiBr/DMF and their molecularweights were analyzed as described earlier. Table 3 shows the effect ofsolvent composition on the polymer molecular weight and dispersity(reactions in bulk and in the absence of reversed micelles). TABLE 3Effect of solvent composition on the polymer molecular weight anddispersity (reactions in the absence of reversed micelles). SynthesisMonomer Polymer Mw/ medium Conversion¹ Yield Mw Mn Comments 100/0  0% 0% no reaction DMF/water 85/15 20% 10% 281 1.23 dimers solubleDMF/water in 85% DMF 60/40 80% 75% 612 1.20 oligomers DMF/water solublein 60% DMF 40/60 80% 80% 675 1.05 oligomers DMF/water soluble in 40% DMF20/80 75% 75% 658 1.02 oligomers DMF/water soluble in 20% DMF 0/100 50%35% 400 1.90 oligomers DMF/water soluble in 100% DMF 85/15 80% 15% 3000 2.10 insoluble Dioxane/water polymer soluble oligomers

[0059] The polymer yield, defined as a ratio of the amount of polymerrecovered as an insoluble fraction in isooctane to the amount of monomerconverted, was about 75% for cases where the DMF content in the reactionmixture was between 20% and 60%. Molecular weight analyses revealed thatthe polymers were in fact oligomers with an average molecular weight ofabout 650, (significantly lower than that obtained with theAOT/isooctane reversed micellar system), and a polydispersity of1.03-1.20. The molecular weight was not variable with DMF content,indicating that either the solubility of growing chains during thereaction was not sustained by DMF/water (up to 60% DMF) or the enzymebecame inactive. Analogous to the dioxane/water system, the monomerconversion was either absent or poor at higher DMF contents, presumablydue to significant enzyme inactivity.

[0060] Ethanol-HEPES buffer mixtures were also used to polymerize m- andp-cresol or p-ethylphenol in the study of molecular weight control.m-Cresol was studied in greater detail since it allows the study of amuch broader molecular weight range than p-cresol or p-ethylphenol.Ethanol is a solvent of choice in view of its environmentalcompatibility and ease of regeneration. In addition, the enzyme isactive in ethanol/buffer mixtures at levels up to at least 60% ethanol.Enzyme activity was studied as a function of ethanol content, and it wasfound that 20 to 40% ethanol was optimal for the conversion of about 50%monomer into polymer (20,000 molecular weight, DP of 200). At an enzymeconcentration of 2 μM, the conversion was essentially complete in about10 minutes. Although there was no significant improvement in the monomerconversion when the reaction was continued for 24 hours, the molecularweight of poly(m-cresol) increased by about 75%. Table 4 shows theeffect of ethanol content on monomer conversion and the molecular weightof poly(m-cresol) in ethanol/water systems. Ethanol appears to be auseful solvent when higher molecular weight polymer is desired.Replacing HEPES buffer with deionized water resulted in no noticeablechange in reaction rates or in polymer properties. TABLE 4 Effect ofethanol content on monomer conversion and the molecular weight ofpoly(m-cresol) in ethanol/water systems. Polydispersity in all caseswas >2.5. Unless noted otherwise, HRP concentration was 0.1 mg/ml.Solvent Monomer Polymer Composition conversion M_(w) Comments 100%buffer 40%  2200 polymer removed once at the end; 60% conversionpossible when HRP and H₂O₂ added in pulses 100% buffer 40%  1400 polymerremoved as it with 1% KCl formed; 60% conversion possible when HRP andH₂O₂ added in pulses 80/20 90% 22000 M_(w) of 6000 to 22000 water/EtOHpossible at intermediate stages of reaction 60/40 47% 10000 Lowermolecular weights water/EtOH possible at intermediate stages of reaction40/60 11%  3000 90% conversion & 24000 M_(w) water/EtOH at 5× enzymeconcentration 20/80  3% — 20% conversion & 2000 M_(w) at water/EtOH 5×enzyme concentration 100% EtOH  0% — Insoluble enzyme

[0061] Although high molecular weight polymers were produced withpoly(m-cresol) due to cross-linking and with ethanol, in someapplications, such as photoresists, oligomers are desirable. Hence,m-cresol was polymerized in 100% buffer/water, and as a result, thepolymer molecular weight decreased to 2,500. During the reaction thepolymer molecular weight gradually increased without significantimprovement in monomer conversion. It was therefore attempted to isolatethe polymer as the polymerization process continued. This was achievedby carrying out the reaction in the presence of 0.5 to 1 (w/v) % KCl inthe medium. While the salt, up to around 3%, had no effect on the enzymeactivity, it caused precipitation of the polymer as it formed. Thepolymer precipitate was isolated by filtration and the filtrate wasreturned to the reaction vessel. The enzyme in the filtrate was nolonger active, therefore, enzyme and peroxide were added in pulses suchthat fresh enzyme was always available after each filtration to assurecontinued polymer formation. However, the rate of polymerization droppedas the reaction continued after each filtration in spite of the presenceof significant amounts of the residual monomer, fresh enzyme, andhydrogen peroxide. The polymer yield was about 40% after completelyadding all the enzyme and hydrogen peroxide to a final concentration of2 μM enzyme and 30% stoichiometric excess of the peroxide. The polymermolecular weight was about 1,400. Further polymerization was stillpossible in the remaining reaction mixture if fresh enzyme and peroxidewere further added. Therefore, the polymerization process could becontinued, in theory, until all monomer was consumed. When the polymerwas not removed by filtration and the reaction was run with theidentical additions of enzyme and hydrogen peroxide, the final polymermolecular weight was over 2,000. The polydispersity was about 2.5 inboth cases of whether the polymer was removed intermittently or not.

[0062] It is thus possible to control the polymer molecular weight bymanipulating the reaction and/or process conditions. In the case ofpoly(m-cresol), it is possible to obtain control of molecular weightfrom 1,400 to 25,000, with a polydispersity of about 2.5, by theselection of the reaction medium. Enzyme activity is a function of thereaction medium composition which influences monomer conversion.However, the polymer molecular weight is also strongly influenced by thepolymer solubility and the length of time it is in contact with theenzyme and reacting species in the reaction mixture, even afterprecipitation.

[0063] In order to minimize the enzyme inactivation, a less polarsolvent than DMF or ethanol was sought as the reaction medium.Accordingly, chloroform was used to carry out the phenol polymerizationreaction. A mixture of chloroform and isooctane was used for thereactions, and the polarity of the medium was gradually varied by addingchloroform and thus changing the composition from 100% isooctane to 100%chloroform. However, the enzyme powder was poorly dispersed in thissolvent system, and it became necessary to prepare AOT reversed micelleswith the chloroform/isooctane mixture, as described earlier. As theisooctane content in the reaction mixture was lowered from 100%, thepolymer yield dropped from 100% (in pure isooctane reversed micelles) toabout 10% (in pure chloroform reversed micelles) These results are shownin Table 5. TABLE 5 Effect of solvent composition on the polymermolecular weight and dispersity (reaction in AOT reversed micelles)using isooctane and chloroform. Synthesis Monomer Polymer mediumConversion Yield Mw Mw/Mn Comments 100% 100% 100% 2500 1.36  W_(o) = 15Isooctane 100%  90% 100% 2500 1.38 W_(o) = 9 Isooctane 75/25 100%  85%1681 1.53 W_(o) = 9 Isooctane/CHCl₃ 50/50 100%  75% 3461 1.85 W_(o) = 9Isooctane/CHCl₃ 25/75  75%  35% 3601 1.83 W_(o) = 9 Isooctane/CHCl₃phase separation 100%  20%  10% 1000 1.07 W_(o) = 9 CHCl₃ phaseseparation

[0064] Polymer molecular weight was maximum at 50-75% chloroform inisooctane with a polydispersity of 1.5 to 1.9. However, the polymerexhibited a low polydispersity of 1.07 in 100% chloroform. The poorpolymer yields at high chloroform contents are perhaps due to theformation of unstable microemulsion systems leading to phase separation.As a result, the contact between the enzyme and the monomer isinefficient and the polymer yield is poor. Smaller W_(o) values alsocontribute to poor monomer conversion. One approach is to eliiminate thesurfactant altogether by polyerizing phenolic monomers in a biphasicsystem where large amount of water containing enzyme is mechanicallydispersed in a hydrophobic organic solvent containing the monomer.Preliminary results indicate that a number of polymers includingpoly(p-ethylphenol) of molecular weight 2500 can be prepared inchloroform/buffer (50:50 v/v) or an isooctane/water (50/50) biphasicsystem.

[0065] The hydroxyl groups in enzymatically prepared polyphenols do nota participate in bond formation, as noted earlier from ¹³C-NMR studies.The FTIR spectrum of the polymer, shown in FIGS. 6a(i) and 6 b(i), alsoillustrates the point with a broad peak at 3400 cm⁻¹ due to O—H stretch.Thus the hydroxyls on the polymer are available for chemicalmodifications such as esterification. Esterification was carried out inchloroform with palmitoyl and cinnamoyl chlorides in the presence ofstoichiometric amount of pyridine to scavenge HCl produced in thereaction.

[0066]FIGS. 6a and 6 b illustrate FTIR spectra of poly(p-ethylphenol)before and after functionalization with palmitoyl and cinnamoylmoieties, respectively, at the hydroxyl groups of the polymer. FIG. 6ashows the presence of alkyl chains in the polymer due to palmitoylgroups, confirmed by the presence of strong peaks between 2800 and 3000cm⁻¹ due to asymmetric and symmetric C—H stretch in methyl and methylenegroups of the alkyl chains.

[0067] In addition, the peak for O—H stretch at 3400 cm⁻¹ disappeared inthe esterified polymer indicating the participation of the hydroxylgroups in the reaction The ester formation was also confirmed by thepresence of C═O stretch at 1750 cm⁻¹ in the modified polymer. Similarly,cinnamoylation of the polymer was confirmed by the disappearance of O—Hstretch as well as from the strong presence of C═C ring stretch at 1600cm⁻¹, shown in FIG. 6b. UV spectroscopic studies, carried out withacetonitrile solutions of the polymer, showed an increased absorbancefor the cinnamoylated polymer at 259 nm due to the presence ofadditional phenyl ring, as shown in FIG. 7.

[0068] It will now be apparent to those skilled in the art that otherembodiments, improvements, details and uses can be made consistent withthe letter and spirit of the foregoing disclosure and within the scopeof this patent, which is limited only by the following claims, construedin accordance with the patent law, including the doctrine ofequivalents.

What is claimed is:
 1. A method of preparing polyphenols andpolyaromatic amines, the method comprising the following steps: (a)preparing a synthesis reaction medium allowing for the control ofmolecular weight and polydispersity of the polymerization product, thesynthesis reaction medium being selected from the group consisting ofreversed micelles, bulk organic solvent mixtures, and biphasic reactionmedia; (b) adding to the synthesis reaction medium an aqueouspreparation of an enzyme; (c) adding to the synthesis reaction mediumand enzyme preparation a monomer selected from the group consisting ofphenols, aromatic amines, and their derivatives to form a reactionmixture; (d) initiating a polymerization reaction by adding dropwise 30%hydrogen peroxide (w/w) (up to about 30% stoichiometric excess) whilestirring the reaction mixture; (e) continuing stirring for severalhours; (f) centrifuging the precipitate formed; (g) repeated washing ofthe precipitate with pure isooctane to remove substances selected fromthe group consisting of the surfactant and any unreacted monomer; and(h) drying the final precipitate overnight under a reduced pressure at50° C.
 2. The method of producing polyphenols and polyaromatic amines,as claimed in claim 1 , wherein the reversed micelles are formed fromdioctyl sodium sulfosuccinate and isooctane.
 3. The method of producingpolyphenols and polyaromatic amines, as claimed in claim 1 , wherein thereversed micelles are formed from chloroform and/or isooctane.
 4. Themethod of producing polyphenols and polyaromatic amines, as claimed inclaim 1 , wherein the bulk organic solvent mixtures are formed fromdimethylformamide and water.
 5. The method of producing polyphenols andpolyaromatic amines, as claimed in claim 1 , wherein the bulk organicsolvent mixtures are formed from dioxane and water.
 6. The method ofproducing poly phenols and polyaromatic amines, as claimed in claim 1 ,wherein the bulk organic solvent mixtures are formed from ethanol andwater.
 7. The method of producing polyphenols and polyaromatic amines,as claimed in claim 1 , wherein the bulk organic solvent mixtures areformed from isooctane and methylchloride.
 8. The method of producingpolyphenols and polyaromatic amines, as claimed in claim 1 , wherein thebiphasic systems are formed from organic solvents and water.
 9. Themethod of producing polyphenols and polyaromatic amines, as claimed inclaim 1 , wherein the enzyme is selected from the group consisting ofperoxidases, tyrosinases, laccases, phenoloxidases, aromaticaminoxidases.
 10. The method of producing polyphenols and polyaromaticamines, as claimed in claim 1 , wherein the monomer is p-ethylphenol.11. The method of producing polyphenols and polyaromatic amines, asclaimed in claim 1 , wherein the monomer is m-cresol.
 12. The method ofproducing polyphenols and polyaromatic amines, as claimed in claim 1 ,wherein the molecular weight of the polymer molecules is between 600 and3,600.
 13. The method of producing polyphenols and polyaromatic amines,as claimed in claim 1 , wherein the molecular weight of the polymermolecules is between 1,400 and 25,000.
 14. The method of producingpolyphenols and polyaromatic amines, as claimed in claim 1 , wherein thepolydispersity of the polymer molecules ranges from 1.02 to greater than2.
 15. A method of producing derivatives of polyphenols and polyaromaticamines, the method comprising the following steps: (a) preparing asynthesis reaction medium allowing for the control of molecular weightand polydispersity of the polymerization product, the synthesis reactionmedium being selected from the group consisting of reversed micelles,bulck organic solvent mixtures, and biphasic reaction media; (b) addingto the synthesis reaction medium an aqueous preparation of an enzyme;(c) adding to the synthesis reaction medium and enzyme preparation amonomer selected from the group consisting of phenols, aromatic amines,and their derivatives, and to form a reaction mixture; (d) initiating apolymerization reaction by adding dropwise 30% hydrogen peroxide (w/w)(up to about 30% stoichiometric excess) while stirring the reactionmixture; (e) continuing stirring for several hours; (f) centrifuging theprecipitate formed; (g) repeated washing of the precipitate with pureisooctane to remove the surfactant and/or any unreacted monomer; and (h)drying the final precipitate overnight under a reduced pressure at 50°C.
 16. The method of producing derivatives of polyphenols andpolyaromatic amines, as claimed in claim 15 , wherein the derivativecomprises palmitoyl chloride.
 17. The method of producing derivatives ofpolyphenols and polyaromatic amines, as claimed in claim 15 , whereinthe derivative comprises cinnamoyl chloride.
 18. The method of producingderivatives of polyphenols and polyaromatic amines, as claimed in claim15 , wherein the derivative comprises biotin compounds.
 19. Apolymerization product produced by: (a) preparing a synthesis reactionmedium allowing for the control of molecular weight and polydispersityof the polymerization product, the synthesis reaction medium beingselected from the group consisting of reversed micelles, bulk organicsolvent mixtures, and biphasic systems; (b) adding to the synthesisreaction medium an aqueous preparation of an enzyme; (c) adding to thesynthesis reaction medium and enzyme preparation a monomer selected fromthe group consisting of phenols, aromatic amines, and their derivativesto form a reaction mixture; (d) initiating a polymerization reaction byadding dropwise 30% hydrogen peroxide (w/w) (up to about 30%stoichiometric excess) while stirring the reaction mixture; (e)continuing stirring for several hours; (f) centrifuging the precipitateformed; (g) repeated washing of the precipitate with pure isooctane toremove the surfactant and any unreacted monomer; and (h) drying thefinal precipitate overnight under a reduced pressure at 50° C.
 20. Thepolymerization product, as claimed in claim 19 , wherein the reversedmicelles are formed from dioctyl sodium sulfosuccinate and isooctane.21. The polymerization product, as claimed in claim 19 , wherein thereversed micelles are formed from AOT, chloroform, and isooctane. 22.The polymerization product, as claimed in claim 19 , wherein the bulkorganic solvent mixtures are formed from dimethylformamide and water.23. The polymerization product, as claimed in claim 19 , wherein thebulk organic solvent mixtures are formed from dioxane and water.
 24. Thepolymerization product, as claimed in claim 19 , wherein the bulkorganic solvent mixtures are formed from ethanol and water.
 25. Thepolymerization product, as claimed in claim 19 , wherein the bulkorganic solvent mixtures are formed from isooctane and methylchloride.26. The polymerization product, as claimed in claim 19 , wherein thebiphasic systems are formed from organic solvents and water.
 27. Thepolymerization product, as claimed in claim 19 , wherein the enzyme isselected from the group consisting of peroxidases, tyrosinases,laccases, phenoloxidases, aromatic aminoxidases.
 28. The polymerizationproduct, as claimed in claim 19 , wherein the monomer is p-ethylphenol.29. The polymerization product, as claimed in claim 19 , wherein themonomer is m-cresol.
 30. The polymerization product, as claimed in claim19 , wherein the molecular weight of the polymer molecules is between600 and 3,600.
 31. The polymerization product, as claimed in claim 19 ,wherein the molecular weight of the polymer molecules is between 1,400and 25,000.
 32. The polymerization product, as claimed in claim 19 ,wherein the polydispersity of the polymer molecules ranges from 1.02 togreater than
 2. 33. A film comprising a polymerization product producedby: (a) preparing a synthesis reaction medium allowing for the controlof molecular weight and polydispersity of the polymerization product,the synthesis reaction medium being selected from the group consistingof reversed micelles, bulk organic solvent mixtures, and biphasicsystems; (b) adding to the synthesis reaction medium an aqueouspreparation of an enzyme; (c) adding to the synthesis reaction mediumand enzyme preparation a monomer selected from the group consisting ofphenols, aromatic amines, and their derivatives to form a reactionmixture; (d) initiating a polymerization reaction by adding dropwise 30%hydrogen peroxide (w/w) (up to about 30% stoichiometric excess) whilestirring the reaction mixture; (e) continuing stirring for severalhours; (f) centrifuging the precipitate formed; (g) repeated washing ofthe precipitate with pure isooctane to remove the surfactant and anyunreacted monomer; and (h) drying the final precipitate overnight undera reduced pressure at 50° C.
 34. A photoresist material comprising apolymerization product produced by: (a) preparing a synthesis reactionmedium allowing for the control of molecular weight and polydispersityof the polymerization product, the synthesis reaction medium beingselected from the group consisting of reversed micelles, bulk organicsolvent mixtures, and biphasic systems; (b) adding to the synthesisreaction medium an aqueous preparation of an enzyme; (c) adding to thesynthesis reaction medium and enzyme preparation a monomer selected fromthe group consisting of phenols, aromatic amines, and their derivativesto form a reaction mixture; (d) initiating a polymerization reaction byadding dropwise 30% hydrogen peroxide (w/w) (up to about 30%stoichiometric excess) while stirring the reaction mixture; (e)continuing stirring for several hours; (f) centrifuging the precipitateformed; (g) repeated washing of the precipitate with pure isooctane toremove the surfactant and any unreacted monomer; and (h) drying thefinal precipitate overnight under a reduced pressure at 50° C.
 35. UVabsorbing materials comprising a polymerization product produced by: (a)preparing a synthesis reaction medium allowing for the control ofmolecular weight and polydispersity of the polymerization product, thesynthesis reaction medium being selected from the group consisting ofreversed micelles and bulk organic solvent mixtures; (b) adding to thesynthesis reaction medium an aqueous preparation of an enzyme; (c)adding to the synthesis reaction medium and enzyme preparation a monomerselected from the group consisting of phenols, aromatic amines, andtheir derivatives to form a reaction mixture; (d) initiating apolymerization reaction by adding dropwise 30% hydrogen peroxide (w/w)(up to about 30% stoichiometric excess) while stirring the reactionmixture; (e) continuing stirring for several hours; (f) centrifuging theprecipitate formed; (g) repeated washing of the precipitate with pureisooctane to remove the surfactant and any unreacted monomer; and (h)drying the final precipitate overnight under a reduced pressure at 50°C.